Preparation of tetrahydrofolic acid from folic acid

ABSTRACT

Improved methods for the preparation and purification of citrovorum factor are disclosed. The method includes improved procedures for hydrogenation of 10-formylfolic acid as well as for the reduction of folic acid. Also disclosed are improved procedures for opening of the imidazoline ring, and a non-chromatographic method for the purification of crude samples of citrovorum factor.

This is a divisional of application Ser. No. 826,677, filed Aug. 22,1977, U.S. Pat. No. 4,148,899.

BACKGROUND AND SUMMARY OF THE INVENTION

The use of high-dose methotrexate therapy with citrovorum factor,5-formyl-5,6,7,8-tetrahydrofolic acid, (5-CHO-THF) rescue is underactive investigation for the treatment of a number of solid tumors andhematologic malignancies. The development of successful protocols willresult in the need for larger amounts of citrovorum factor (5-CHO-THF).

By the present invention, there are provided improved methods for thepreparation and purification of citrovorum factor. In one aspect of theinvention, formylation of folic acid (FA) gave 10-CHO-FA, which washydrogenated in trifluoroacetic acid to give high yields of (5,10-CH-THF)⁺, the dehydration product of the initially formed 10-CHO-THF.In another aspect of the invention, the reduction of folic acid withborohydride followed by treatment of the resulting THF with formic acidgave good yields of (5, 10-CH-THF)⁺, isolated as the chloride. Theeffect of base concentration, temperature, and time of reaction on theconversion of (5, 10-CH-THF)⁺ CL⁻ to 5-CHO-THF was determined. Thesemethods led to the preparation of the calcium salt dihydrate of5-CHO-THF in high yields, which was about 78% pure. The identificationof the impurities in these 5-CHO-THF samples was determined byhigh-pressure liquid chromatography, and the removal of the impuritieswas effected by Florisil chromatography. The discovery of anonchromatographic method for the removal of most of the impurities fromcrude samples of 5-CHO-THF is also described.

The decrease in toxicity and increase in therapeutic benefit resultingfrom the adjuvant treatment of osteogenic sarcoma with a high dose ofmethotrexate followed by rescue with citrovorum factor,5-formyl-5,6,7,8-tetrahydrofolic acid, (5-CHO-THF) has been establishedby Jaffe et al., New Engl. J. Med., 291, 994 (1974). In this modality,5-CHO-THF apparently protects normal sensitive tissue without cancelingthe inhibitory activity of methotrexate against neoplastic tissue. Inaddition, this form of therapy has been reported as being potentiallyeffective against other malignancies, including refractory acuteleukemia, bronchogenic carcinoma and head and neck cancer. Although theultimate value in terms of cures of this form of therapy has not beenfully documented, the development of the high-dose methotrexate regimenrequires large amounts of both methotrexate and 5-CHO-THF. Recently,there has been reported by J. R. Piper and J. A. Montgomery, J.Heterocycl. Chem., 11, 279 (1974), an improved method for thelarge-scale synthesis of methotrexate of high purity, and we now reportimproved procedures for the large-scale preparation and purification of5-CHO-THF.

The synthesis and identification of 5-CHO-THF was carried out about 25years ago, as described, for example, by Pohland et al., J. Am. Chem.Soc., 73, 3247 (1951). In general, the adopted procedure involved theformylation of folic acid with formic acid, catalytic hydrogenation ofthe pyrazine ring of the resulting formic acid solution of 10-CHO-FA,and treatment of the product of the reduction with base at elevatedtemperatures to give crude 5-CHO-THF. In one procedure, bioassay of thecrude product indicated that about a 22% yield of 5-CHO-THF wasobtained. Purification was effected by column chromatography to give alow yield of 5-CHO-THF isolated as the barium salt pentahydrate.Although no yields were reported,, a similar procedure was used for thepreparation of the calcium salt of 5-CHO-THF.

In the prior art synthesis described above, it was recognized that5-CHO-THF was dehydrated under acidic conditions to give (5, 10-CH-THF)⁺and that the same product was formed from 10-CHO-THF resulting from thehydrogenation of 10-CHO-FA in formic acid. Treatment of (5, 10-CH-THF)⁺with base at room temperature opened the imidazolinium ring to givemainly 10-CHO-THF (kinetic control), which underwent oxidation readilyin the presence of oxygen and light to give decomposition products, asdescribed by May et al., J. Am. Chem. Soc., 73, 3067 (1951). Incontrast, treatment of (5,10-CH-THF)⁺ with base at higher temperaturesresulted in the formation of 5-CHO-THF (thermodynamic control) anddecomposition products.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

In the procedures according to the present invention, the first approachwhich was carried out included treatment of folic acid with 98% HCO₂ Hunder N₂ at 60° to give 10-CHO-FA, which was purified byrecrystallization from H₂ O. All temperatures as stated herein are indegrees Centigrade. As was noted in the Pohland et al reference citedabove, considerable difficulty was encountered in the hydrogenation of10-CHO-FA in HCO₂ H, with this reduction requiring more than 40 hr forthe uptake of 2 molar equivalents of H₂ in the presence of platinum atroom temperature and atmospheric pressure. Because of these results,conditions were sought for the reduction of 10-CHO-FA to give10-CHO-THF, which might undergo in situ rearrangement to give 5-CHO-THF.

In the hydrogenation of 10-CHO-FA in pyridine in the presence of Pd, 2molar equivalents of H₂ appeared to be absorbed within 5 hr; however,the recovery of 10-CHO-FA from this reaction indicated thathydrogenation of the solvent rather than the pyrazine ring of 10-CHO-FAhad occurred. Based on the successful chemical reduction of folic acidto THF with Na₂ S₂ O₄, the reduction of 10-CHO-FA with this reagent wasattempted. However, treatment of solutions of 10-CHO-FA in aqueouspyridine and in water at pH 6, 7, and 8.5 with Na₂ S₂ O₄ at 75° resultedin the formation of complex mixtures (TLC), which were not examinedfurther. In addition, treatment of 10-CHO-FA either at room temperaturewith excess aqueous NaBH₄ (alkaline medium) or with refluxing aqueousNaBH₃ CN at pH 6.7 resulted in extensive decomposition of the samplewith little or no conversion to 5-CHO-THF. Although treatment of10-CHO-FA with an equal weight of NaBH₄ while maintaining the pH of themedium near 8 with HOAc followed by refluxing the reaction mixture afteradjustment to pH 6.7, as described hereinbelow, gave a mixturecontaining 5-CHO-THF, the mixture contained a large amount of unreduced10-CHO-FA. These difficulties were circumvented by the catalytichydrogenation of 10-CHO-FA in CF₃ CO₂ H containing prereduced PtO₂,which was more than 20 times faster than the hydrogenation of 10-CHO-FAin HCO₂ H.

Next, the formylation and hydrogenation of folic acid without isolationof 10-CHO-FA was carried out. The solution of 10-CHO-FA in HCO₂ Hresulting from the formylation of folic acid was diluted with an equalvolume of CF₃ CO₂ H and hydrogenated in the presence of prereduced PtO₂(20% by weight of folic acid). The absorption of H₂ was slower thanexpected, and the reaction was repeated by removing the formic acidafter formylation and using a smaller amount of catalyst, 5% by weightof folic acid. Under these conditions, the catalyst appeared to bepoisoned after the uptake of 1 molar equivalent of H₂, but the reductionreturned to the original rate after the addition of either freshlyreduced PtO₂ (total 7.5% by weight of folic acid) or additional solvent.Based on these results, large-scale preparations were carried out by theformylation of folic acid with HCO₂ H, recovering the HCO₂ H bydistillation in vacuo, and hydrogenation of the resulting dried residuein CF₃ CO₂ H containing prereduced PtO₂ (5% by weight of folic acid) atroom temperature and atmospheric pressure. Although it was found thatCF₃ CO₂ H was reduced by Pt, the reaction was slower than the reductionof 10-CHO-FA. In addition, none of the reduction products from CF₃ CO₂ Happeared to interfere either with the hydrogenation of 10-CHO-FA or theisolation of its reduction product. Both the solvent and catalyst couldbe recovered and reused at least five times.

Although the hydrogenation of 10-CHO-FA in CF₃ CO₂ H probably gave10-CHO-THF initially, this compound was readily dehydrated by thesolvent. The resulting product was an acylated derivative (either formylor trifluoroacetyl or both) of (5, 10-CH-THF)⁺, isolated as a foam,which was converted by dissolution in 0.5 N HCl and concentration of theresulting solution to give a precipitate of practically pure (5,10-CH-THF)⁺ Cl⁻. The obtainment of this product in an amount equal tothe weight of the folic acid starting material indicated almostquantitative conversion in each step of the reaction sequence.

As a variation of the above approach, the large-scale reduction of folicacid to THF was investigated. Previously, the reduction of folic acidwith borohydride gave a mixture of unreacted folic acid, DHF, and THF,the latter in yields up to 50%. Preliminary experiments indicated thatan amount of sodium borohydride equal to the weight of folic acid wasnecessary to ensure complete reduction. To circumvent the use of abuffered medium, the reduction was carried out by the portionwiseaddition of an aqueous solution of NaBH₄ while maintaining the pH of thereaction medium near 8 by the addition of dilute HCl. However, someoxidation of the product occurred upon acidification of the reactionmixture to precipitate THF. Additional experiments revealed that theaddition of HCl during the reduction was unnecessary as the systembecame buffered with borate at a pH of less than 10. The reoxidation ofthe THF during the isolation prevented by the addition of ascorbic acidduring the acidification, which gave THF as a boron complex in 96%yield.

The direct conversion of the free acid of THF to 5-CHO-THF was effectedwith a refluxing 95:5 mixture of pyridine-HCO₂ H containing ascorbicacid. However, after a basic workup, the isolated CF contained aconsiderable amount of 10-CHO-DHF (TLC,HPLC), and no further work wascarried out on this method.

Treatment of the THF product with either HCO₂ H or 2:1 HCO₂ H-CF₃ CO₂ Hat room temperature gave mainly (5,10-CH-THF)⁺. Similarly, the sameproduct resulted from the condensation of THF with (EtO)₃ CH either inthe presence of HCl at room temperature or in the presence of aqueousascorbic acid at reflux. The yields and purity of the product appearedto be better in the procedures using HCO₂ H, and the adopted procedureinvolved the dissolution of the precipitated THF directly in 98:2 HCO₂H-CF₃ CO₂ H, which provided (5,10-CH-THF)⁺ free of boron impurities.This procedure was improved by elimination of the step involving theisolation of THF. Treatment of the aqueous solution (pH 7) of THFresulting from the NaBH₄ reduction of FA with an equal volume of HCO₂ Hresulted in the isolation of a good yield of (5,10-CH-THF)⁺ CL⁻.

On TLC many of these samples of (5,10-CH-THF)⁺ Cl⁻ exhibited traceamounts of colored fluorescent impurities, which were removed by columnchromatography. This imidazolinium chloride appeared to be stable in thesolid state and in acidified aqueous solution, but, as noted above, wasunstable in the presence of aqueous base. Additional information on thetransformation of (5,10-CH-THF)⁺ Cl⁻ in solutions was provided by the uvspectrum of the eluted peaks obtained on the chromatograms produced byhigh-pressure liquid reverse-phase chromatography.

When (5,10-CH-THF)⁺ Cl⁻ was dissolved in pH 5 buffer, a mixture of10-CHO-DHF, (5,10-CH-THF)⁺, and 5-CHO-THF was formed. Presumably,(5,10-CH-THF)⁺ was converted to 5,10-(HOCH)-THF, which underwentdifferent modes of ring opening to give a small amount of 5-CHO-THF anda large amount of 10-CHO-THF followed by air oxidation of the latter togive 10-CHO-DHF. No 10-CHO-DHF was formed when (5,10-CH-THF)⁺ wasdissolved in pH 5 buffer containing ascorbic acid, the resultingsolution exhibiting peaks only for (5,10-CH-THF)⁺ and 5-CHO-THF. Incontrast, the chromatogram of a solution of (5,10-CH-THF)⁺ at pH 7 inthe presence of ascorbic acid showed only a small amount of 10-CHO-DHFand two new peaks, which were tentatively assigned to 10-CHO-THF and5,10-(HOCH)-THF. A later chromatogram of this solution showed that bothof the peaks assigned to 10-CHO-THF and 5,10-(HOCH)-THF decreased withtime while the 10-CHO-DHF peak increased even in the presence ofascorbic acid. The possibility was not eliminated from considerationthat the peak assigned to 5,10-(HOCH)-THF was a tautomeric form of10-CHO-DHF formed initially in the oxidation of 10-CHO-THF.

A solution of (5,10-CH-THF)⁺ Cl⁻ at pH 13 containing ascorbic acidshowed only the peak assigned to 10-CHO-THF. Although the uv spectrum ofthis peak was similar to that of 10-CHO-DHF, this resulted from10-CHO-THF being converted rapidly to 10-CHO-DHF in the uv cell by uvradiation. This transformation was followed by measuring the increase inabsorbance at a wavelength (330 nm) where 10-CHO-DHF gave a maximumafter the peak was eluted from the column. Surprisingly, treatment of(5,10-CH-THF)⁺ Cl⁻ with base at pH 11 with no protection from O₂ gave ahigh yield of 10-CHO-THF, which was isolated as its calcium salt. Thestructure of the latter was confirmed by its reconversion to(5,10-CH-THF)⁺ Cl⁻ in an acidic medium (uv). Further treatment of10-CHO-THF in a basic medium, however, resulted in the formation of amixture of 10-CHO-DHF, 10-CHO-FA, and decomposition products. Theobtainment of a purified sample of 10-CHO-DHF by elution of this mixturefrom a Florisil column with 0.1 M mercaptoethanol was unsuccessfulbecause most of the sample decomposed on the column. Not only was theweight of the recovered material low (15%), but HPLC showed that the10-CHO-DHF obtained was contaminated with p-aminobenzoylglutamic acid,pterins, and unidentified substances.

Previously, the (5,10-CH-THF)⁺ prepared in situ was converted to5-CHO-THF in a hot, neutral or alkaline medium with a reaction time ofabout 1 hour. The solid (5,10-CH-THF)⁺ Cl⁻ prepared above was used insmall-scale experiments to determine the effect of base concentration,temperature, and time of reaction on the purity of the 5-CHO-THFobtained. The progress of the reaction was followed by the determinationof the uv spectrum of aliquot portions in 0.1 N NaOH and comparison ofthe λ_(max) ²⁸² /λ_(min) ²⁴² ratio with that observed in the isolatedsample of 5-CHO-THF. The uv data indicated that the ratio increasedfaster at the higher pH values, but also indicated that the ratioreached a maximum and then decreased. In addition, carrying out thereaction in a pressure apparatus at higher temperatures increased therate of formation of citrovorum factor, but offered no advantage inregard to the purity of the product.

In one reaction in which the initial pH was 8.3 and the final pH 5.9,the same ratio was observed for the last aliquot portion and theisolated product suggesting that the reaction mixture was atequilibrium. In this reaction, a 75% increase in absorbancy at 282 nmoccurred within 2.5 hours followed by a smaller increase over theremaining time of the experiment. A log A vs. time plot suggested thatthe conversion involved two sequential first-order reactions: possiblythe transformation of (5,10-CH-THF)⁺ via 5,10-(HOCH)-THF to 5-CHO-THFand 10-CHO-THF followed by the reversible transformation of 10-CHO-THFvia 5,10-(HOCH)-THF to 5-CHO-THF, a reaction that would be expected topredominate in the latter part of the conversion.

A highly significant result of this study was the discovery that therate of opening of the imidazolinium ring of (5,10-CH-THF)⁺ wasreasonable and that the purity of the 5-CHO-THF formed was greatest whenthe reaction was performed under neutral or slightly acidic conditions,at a pH of about 6.2 to 7.0. The latter result was confirmed inreactions with (5,10-CH-THF)⁺ at pH 11.4, 9, and 6.2 to give 5-CHO-THFof increasingly greater purity. However, experiments carried out indilute solutions of 5-CHO-THF at 100° for 7 hours over the pH range6.5-5.5 showed that increasing amounts of 10-CHO-DHF, 10-CHO-FA, andp-aminobenzoylglutamic acid (PABGA) were formed as the pH was decreased.For the large-scale synthesis of 5-CHO-THF from (5,10-CH-THF)⁺ Cl⁻ inconcentrated solutions, the pH of the reaction medium was maintainednear 6.7 for about 11 hours, which gave a product with a uv ratio of3.4-3.9. These samples of 5-CHO-THF contained 10-CHO-DHF and PABGA asthe major impurities and 10-CHO-FA and pterins as minor impurities. OnTLC (5,10-CH-THF)⁺, 10-CHO-DHF, and 10-CHO-FA were detectable at lessthan 5% of the concentration of 5-CHO-THF. The major portion of the10-CHO-DHF impurity in 5-CHO-THF samples was formed from unconverted(5,10-CH-THF)⁺ and 10-CHO-THF during the basic workup of the reaction.

The calcium salt of 5-CHO-THF appeared to have little or no solubilityin anhydrous organic solvents; in fact, the salt was reprecipitated froman aqueous solution on the addition of DMAC. Also, recrystallization ofthe salt from a saturated solution of CaCl₂ and fractional precipitationof the salt from water-ethanol mixtures was unsatisfactory for thepreparation of purified samples. In addition, the preparation of a PG,17purified sample of 5-CHO-THF by column chromatography was unsuccessfulwith the following packings: cation spherical resin developed with 0.1 MCaCl₂ -Ca(OH)₂ (pH 10); cation exchange cellulose developed with 0.1 MCaCl₂ -HCl (pH 5.5); Avicel cellulose developed with 0.1 M CaCl₂ (pH 8);silica gel H developed with 7:3 H₂ O-acetone under water pressure;polyethylene powder developed with 3:1 H₂ O-acetone; and Sephadex G-75developed with H₂ O (pH 8). Practically pure 5-CHO-THF was obtained fromcrude samples by column chromatography on Sephadex G-10 developed withaqueous Ca(OH)₂ (pH 8), as further described hereinafter. A bettermethod, however, was the column chromatography of crude samples onFlorisil developed with aqueous mercaptoethanol, which gave 5-CHO-THF in28-35% yield (from FA). These samples were shown by HPLC to contain onlytrace amounts of uv-absorbing impurities.

As a further aspect of the present invention, a nonchromatographicprocedure was developed for the removal of most of the impurities fromcrude 5-CHO-THF samples. A solution of the sample in water containingmagnesium chloride was adjusted to pH 12 with calcium hydroxide toproduce a precipitate of the inorganic oxides, most of the impurities,and some 5-CHO-THF. From the filtrate 5-CHO-THF of good quality wasrecovered. This method is potentially the most convenient procedure forthe purification of 5-CHO-THF.

The diagram shows the various reactions which take place in accordancewith the present invention.

EXAMPLE 1 10-Formylfolic Acid (10-CHO-FA)

A mixture of FA.2H₂ O (30.0 g. 62.8 mmol) and 98% HCO₂ H (400 ml) wasstirred under N₂ in a 60° oil bath for 2 hours. The resulting solutionwas evaporated to dryness under reduced pressure, and the residue wasdried in vacuo over P₂ O₅ and NaOH pellets for 18 hours to give a dry,glassy material: yield, 37.2 g. λ_(max), nm: 0.1 N HCl-252, 322; pH7-260, 349; 0.1 N NaOH-257, 365. Pmr (DMSO-d₆, 5.5% g/ml)- δ8.14 (CHOmoiety, position unidentified), 8.63 (7-CH), 8.79 (10-CHO). TLC [Avicel,0.1 M phosphate buffer (pH 7)]-Rf˜0.85 (fluorescent).

In a 1.0-g run the isolated residue was recrystallized from hot H₂ O(110 ml) to give the monohydrate: yield, 0.57 g (56%). This sampleunderwent decomposition from about 200°. λ_(max), nm (ε×10⁻³): 0.1 NHCl-252 (26.5), 322 (8.69); pH 7-249 sh (24.3), 263 br (25.6), 348(6.15), 357 sh (5.81); 0.1 N NaOH-257 (39.0), 367 (7.62). ν_(max), cm⁻¹: 1680 br. Pmr (DMSO-d₆, 4.3% g/ml)-δ8.63 (7-CH), 8.79 (10-CHO).##STR1##

Anal. Calcd for C₂₀ H₁₉ N₇ O₇. H₂ O: C, 49.28; H, 4.34; N, 20.12. Found:C, 49.12; H, 4.27; N, 19.97.

EXAMPLE 2 Calcium 10-Formyl-7,8-dihydrofolate

A suspension of (5,10-CH-THF)⁺ Cl⁻ (10.0 g) in H₂ O (315 ml) was stirredand treated with 1 N NaOH (˜85 ml) to give a clear yellow solution (pH11, meter). After stirring at room temperature with free access to airfor 2 hours, the solution was adjusted to pH 7.5 with dilute HClfollowed by the addition of a clarified solution of CaCl₂ (2.5 g/5 ml)and EtOH (150 ml). The yellow precipitate (0.6 g) that deposited wasremoved by filtration, and the filtrate was diluted with an additionalamount of EtOH (800 ml). The resulting pale yellow precipitate wascollected by filtration and dried in vacuo over P₂ O₅ : yield, 9.74 g.The uv spectrum indicated that this solid was mainly calcium10-formyl-5,6,7,8-tetrahydrofolate. λ_(max), nm (ε×10⁻³): pH 7 (1%mercaptoethanol)-257 (18.8), 305 sh (6.12), 340 sh (2.85). After 24hours this solution gave the following spectrum: 262 (17.5), 305 sh(7.56), 340 sh (3.60).

Anal. Calcd for C₂₀ H₂₁ N₇ O₇.Ca .0.75 C₂ H₆ O.2H₂ O: C, 44.36; H, 5.11;N, 16.84; Ca, 6.89; Ash (CaO), 9.65. Found: C, 44.69; H, 4.91; N, 17.06;Ca, 6.82; Ash (CaO), 10.17.

A portion of the above solid (5.0 g) was dissolved in H₂ O (500 ml), andthe solution (pH 7.4) was stirred in the presence of air at roomtemperature for 18 hours. During this period, a yellow solid depositedas the pH of the solution dropped to 6.7. The solid was collected byfiltration and dried in vacuo over P₂ O₅ : yield, 0.76 g. The ¹ H NMRspectrum, elemental analyses, and HPLC assay of this sample showed thatit was a 2:1 mixture of the calcium salts of 10-formyl-7,8-dihydrofolateand 10-formylfolic acid. λ_(max), nm (ε×10⁻³): pH 7-233 (30.8), 260 sh(22.8), 266 sh (22.3), 335 (6.68). TLC [Avicel, 0.1 M phosphate bufferpH 7)]-R_(f) 0.70 (10-CHO-DHF), 0.86 (10-CHO-FA) (both fluorescent).

Anal. Calcd for (C₂₀ H₁₉ N₇ O₇)₂ (C₂₀ H₁₇ N₇ O₇).Ca.2H₂ O: C, 44.09; H,4.13; N, 18.00; Ca, 7.36; Ash (CaO), 10.30. Found: C, 44.18; H, 4.10; N,17.99; Ca, 7.33; Ash (CaO), 9.93.

Dilution of the filtrate from the above solid with EtOH gave a mixtureof the salts of 10-formyl-7,8-dihydro- and 10-formylfolic acids (3.21 g)that was contaminated with other impurities (p-aminobenzoylglutamicacid, pterins).

EXAMPLE 3 5,6,7,8-Tetrahydrofolic Acid

To a suspension of folic acid dihydrate (47.8 g, 100 mmol) in deaeratedH₂ O (1,000 ml), which was cooled in an ice bath, was added slowly withstirring 50% NaOH (10.5 ml). The resulting dark yellow solution (pH 8.0,meter) was treated over a 10-min period with a solution of NaBH₄ (58 g)in H₂ O (150 ml). The solution was stirred for an additional 30 min, thepH increasing during this period from 8.4 to 8.8. Excess NaBH₄ wasdecomposed by the addition of 6 N HCl (caution, vigorous effervescence)until the pH of the solution was 6.8. Solid ascorbic acid (5 g) wasadded and stirring continued until complete dissolution occurred. Theresulting solution was adjusted to pH 3.7 with 6 N HCl (total volume,165 ml). The cream-colored precipitate was collected by filtration underN₂, washed with ice cold HCl (pH 3.5, 200 ml) containing ascorbic acid(2 g) and dried to constant weight in vacuo over P₂ O₅ at roomtemperature: yield, 60.0 g (96%). Solutions for the uv spectradeterminations were obtained by dissolution of tetrahydrofolic acid (5.5mg) by the successive addition of mercaptoethanol (2.5 ml) and water(22.5 ml) and dilution of the resulting stock solution with theappropriate solvent (5→50 ml). λ_(max), nm (ε×10⁻³): 0.1 N HCl-270(23.5), 292 (20.8); pH 7-297 (25.7); 0.1 N NaOH-297 (25.9). TLC [DEAEcellulose; 0.005 M KH₂ PO₄ +0.5 M NaCl+0.2 M mercaptoethanol (pH7)]-R_(f) ˜0.44 (elongated).

This sample analyzed for the following composition:

Anal. Calcd for C₁₉ H₂₃ N₇ O₆.1.66HCl.1.68H₃ BO₃.0.74H₂ O: C, 36.66; H,5.04; B, 2.92; Cl, 9.45; N, 15.75. Found: C, 36.69; H, 5.20; B, 2.89;Cl, 9.45; N, 15.66.

EXAMPLE 4 Calcium Salt of Citrovorum Factor

The combined crops of (5,10-CH-THF)⁺ Cl⁻ (1212 g), prepared as describedbelow, were added with stirring under a N₂ atmosphere to boiling H₂ O(30 l.) over a period of 20 minutes. During the addition and thereafterfor 1 hour, hot, oxygen-free 3.7 N NaOH (˜21.) was added at a rate tomaintain an acidic reaction medium. At this point, complete dissolutionof the solid was obtained with oxygen-free 1 N NaOH. The resultingsolution was refluxed for 11 hours while maintaining the pH between6.5-6.9 (meter) with 1 N NaOH (total,˜450 ml). The progress of thereaction was followed by determining the HPLC chromatograms of aliquotportions. After standing for an additional 8 hours without heat, thesolution (56°, pH 7.7) was treated with a clarified solution (1200 ml)of CaCl₂ (600 g), which lowered the pH to 7.2. The solution was dilutedwith EtOH (3.2 l.) and transferred through tygon tubing with aperistaltic pump to a flask cooled in an ice-salt mixture. When thetemperature of the mixture was less than 10°, the bright yellow solidthat deposited was removed by filtration. On exposure to air, this soliddarkened to a brown color and became gummy. TLC of the semidried residue(˜400 g) showed that it contained 5-CHO-THF contaminated with numerousimpurities. Further work on the characterization of this residue isdescribed in the section of the nonchromatographic purification of5-CHO-THF.

The clear yellow filtrate from above was pumped into a large containerand diluted with EtOH (total, 102 l.). The resulting slurry ofcream-colored precipitate of 5-CHO-THF.Ca was cooled (<10°) in an icebath for 18 hours, the solid was collected by filtration, washed withEtOH (7 l.), and dried in vacuo over P₂ O₅ : yield, 897 g (˜45% fromFA). λ_(max), nm (ε×10⁻³): 0.1 N NaOH-282 (28.8) [λ_(max) ²⁸² /λ_(min)²⁴² (0.1 N NaOH), 3.6]. TLC [Avicel, 0.1 M phosphate buffer (pH7)]-R_(f) 0.76 (10-CHO-DHF), 0.85 (CF, absorbing), 0.93 (10-CHO-FA).

Anal. Calcd for C₂₀ H₂₁ N₇ C₇.Ca.0.5C₂ H₆ O.1.8H₂ O: C, 44.49; H, 4.91;N, 17.29; Ca, 7.07; Ash (CaO), 9.89. Found: C, 44.49; H, 4.97; N, 17.29;Ca, 7.18; Ash (CaO), 9.65.

HPLC assay of this sample indicated the presence of 5-CHO-THF.Ca (78%)and, excluding ethanol and water, the following impurities: PABGA.Ca(3.1%), 10-CHO-DHF (4.6%), 10-CHO-FA (<0.5%), pterins (1.0%), andunidentified and undetected material (3.0%).

EXAMPLE 5 5,10-Methenyl-5,6,7,8-tetrahydrofolic Acid (5,10-CH-THF)⁺ (A).

A suspension of PtO₂ (1.50 g) in CF₃ CO₂ H (600 ml) was hydrogenated at24° and atmospheric pressure until the theoretical volume (322 ml) of H₂was absorbed (<5 min). To this mixture was added a solution of crude10-CHO-FA (37.1 g, from 30 g FA) in CF₃ CO₂ H (900 ml), and the wholewas hydrogenated with rapid magnetic stirring at 24.5° and atmosphericpressure. Within 2.5 hours, the theoretical amount of H₂ was absorbed(3,095 ml). The resulting mixture was filtered (Celite) under N₂pressure, and the filtrate was evaporated at less than 40° under reducedpressure to give a dry, porous foam. After drying this sample for 18hours over P₂ O₅, the foam was dissolved in 0.5 M HCl (200 ml) that was0.1 M in 2-mercaptoethanol. The dark solution was warmed to 40° (H₂ Obath), treated with charcoal (0.5 g), and filtered (Celite). The filterpad was washed with the 0.5 M HCl-0.1 M mercaptoethanol solvent (100ml), and the clear yellow filtrate was concentrated at 40° to 2/3 volumeunder aspirator vacuum. The resulting mixture was cooled; the yellowsolid of the chloride salt was collected by filtration, washed with the0.5 M HCl-0.01 M mercaptoethanol solvent (25 ml), and dried in vacuoover P₂ O₅ and NaOH pellets for 18 hours: yield, 29.9 g. λ_(max), nm: 1N HCl-286, 347 (A_(max) ³⁴⁷ ;A_(min) ³⁰² =2.60; lit. A_(max) ³⁴⁸/A_(min) ³⁰⁵ =2.46). ν_(max), cm⁻¹ : 1730, 1655 sh, 1630, 1620 sh. Pmr(CF₃ CO₂ D, 5.3% g/ml), δ9.57 (methenyl CH). TLC [Avicel, 0.1 Mphosphate buffer (pH 7)] showed an elongated bluish-white fluorescentspot at R_(f) 0.47 and a yellow fluorescent impurity spot near theorigin. Concentration of the filtrate to about 20 ml from the first cropgave a dark colored solid: yield, 0.9 g. TLC indicated that this samplecontained more of the yellow fluorescent impurity. In this hydrogenationboth the catalyst and solvent could be recovered and reused at least 5times.

The yellow fluorescent impurity in crude (5,10-CH-THF)⁺ Cl⁻ from anotherrun was removed by column chromatography. This sample (0.50 g) waseluted at a rate of 6 ml/10 min from an Avicel (75 g) column with HCl(pH 2.3) that was 0.01 M in 2-mercaptoethanol. Fractions 20-25, whichcontained crystalline (5,10-CH-THF)⁺ Cl⁻, were combined and concentratedin vacuo below room temperature to a thick slurry. The yellow solid wascollected by filtration under N₂, washed with EtOH and Et₂ O, and driedin vacuo over P₂ O₅ for 18 hours: yield, 0.24 g (48% recovery). λ_(max),nm (ε×10⁻³): 1 N HCl-286 (12.4), 347 (25.8) [A_(max) ³⁴⁷ /A_(min) ³⁰³=2.57]. ν_(max), cm⁻¹ : 1730, 1660 sh, 1630 br. Pmr (CF₃ CO₂ D, 5.3%g/ml), 9.56 (methenyl CH). TLC [DEAE cellulose; 0.005 M phosphatebuffer, 0.5 M NaCl, 0.2 M mercaptoethanol (pH 7)] showed one major spotand a trace amount of a lower absorbing impurity spot.

Anal. Calcd for C₂₀ H₂₂ ClN₇ O₆.0.1HCl.H₂ O: C, 46.78; H, 4.73; Cl,7.59; N, 19.09. Found: C, 46.87; H, 4.79; Cl, 7.50; N, 18.99.

Fractions 26-42 from the above column were treated in the same manner:yield, 0.14 g (28% recovery). λ_(max), nm: 1 N HCl-286, 347 [A_(max) ³⁴⁷/A_(min) ³⁰³ =2.62]. TLC of this sample was similar to the first crop.The total amount recovered was 0.38 g (76%).

In another run a sample of crude (5,10-CH-THF)⁺ Cl⁻ (100 mg) was elutedat a rate of 4 drops/min from an Avicel (15 g) column (1×55 cm) with 0.1M HC0₂ -H-0.01 M mercaptoethanol. Twelve fractions (5 ml each) werecollected in which fractions 5-7 deposited crystalline product. Thesefractions were combined and cooled; the solid was collected byfiltration, washed with 0.1 M HCO₂ H-0.01 M mercaptoethanol, and driedin vacuo over P₂ O₅ : yield, 12 mg (12% recovery). This material washomogeneous on TLC (DEAE cellulose). The mother liquor from this sampleand fractions 4 and 8-12 were combined and evaporated to dryness. Theresulting residue (70 mg) was recrystallized from 0.1 M HCO₂ H-0.01 Mmercaptoethanol: yield, 35 mg (35% recovery). λ_(max), nm (ε×10⁻³): 1 NHCl-286 (12.1), 347 (25.5) [A_(max) ³⁴⁷ /A_(min) ³⁰³ =2.60]. Thismaterial was homogeneous on TLC; however, elemental analyses indicatedthat this material was a mixture of the chloride (34%) and thecorresponding meso-ionic compound (66%).

Anal. Calcd for [34% [(C₂₀ H₂₂ N₇ O₆)⁺ Cl⁻ ].66% (C₂₀ H₂₁ N₇ O₆)].3H₂ O:C, 46.03; H, 5.28; Cl, 2.31; N, 18.79. Found: C, 46.00; H, 5.20; Cl,2.37; N, 18.57.

(B) To a suspension of FA.2H₂ O (1673 g, 3.500 mol) in H₂ O (35 l.),which was under an atmosphere of N₂ and cooled to 8° in an ice bath, wasadded slowly with stirring 50% NaOH (370 ml). The resulting clear yellowsolution (pH 8, meter) was treated over a one-hour period with asolution of NaBH₄ (1673 g) in H₂ O (5 l.). During the addition, thetemperature increased to a maximum of 17°. The solution was stirred foran additional 30 minutes, followed by the decomposition of excess NaBH₄with concd HCl. The large amount of H₂ generated was vented to a hood.During the first half of the decomposition step, efficient cooling wasrequired to maintain the temperature of the solution below 24°. Thedecomposition of NaBH₄ was essentially complete after the addition of2000 ml of concd HCl, which required a period of 3 hours. The resultingsolution (pH 8.3) was adjusted to pH 6.6 with 500 ml of concd HCl over aperiod of 30 minutes. At this point, a solution of ascorbic acid (175 g)in H₂ O (800 ml) was added to protect THF against air oxidation. The pHof the solution was then adjusted to 3.5 with an additional 1800 ml ofconcentrated HCl over a period of one hour. The resulting cream-coloredsuspension of THF was pumped into a Buchner funnel (11-l. capacity)fitted with a glass fiber paper (Whatman GF/D) and under an atmosphereof N₂. This filtration (aspirator pressure) was carried out in twobatches because of the large amount of solid. Near the end of thefiltration, a small portion (˜50 g) of the THF slurry was exposed to airand was discarded. Each batch of the wet precipitate was dissolved in amixture of 98:2 HCO₂ H (97%)-CF₃ CO₂ H and transferred under aspiratedvacuum to a 24-l. flask. A total volume of 12,750 ml of the acid mixturewas used. After standing at room temperature for 14 hours, the dark redsolution was evaporated to dryness in vacuo at a maximum H₂ O-bathtemperature of 60°. The superficially dried residue was suspended in 0.5N HCl (35 l.) containing 2-mercaptoethanol (1 ml/l. of acid), warmed to45°, and the whole was concentrated under aspirator pressure to removeformic and trifluoroacetic acids (˜3 l.). After standing at roomtemperature for 18 hours, the (5,10-CH-THF)⁺ Cl⁻ was collected byfiltration on a glass fiber paper, washed with 0.01 N HCl (6 l.), anddried in vacuo over P₂ O₅ : yield, 1119 g (63%). A boron analysisindicated the absence of boron salts.

Anal. Calcd for (C₂₀ H₂₂ N₇ O₆)⁺ Cl⁻.H₂ O: C, 47.11; H, 4.74; Cl, 6.75;N, 19.23. Found: C, 47.24; H, 4.65; Cl, 7.16; N, 19.28.

Concentration of the filtrate to about one-third the original volumedeposited a second crop, which was less pure (5,10-CH-THF)⁺ Cl⁻ : yield,95 g (˜5%). The total yield was 1214 g (˜68%).

Modification of the procedure described above gave a higher yield of(5,10-CH-THF)⁺ Cl⁻. FA.2H₂ O (10.0 g, 20.9 mmol) was treated with NaBH₄,the excess NaBH₄ was decomposed, and the resulting solution (pH 7) wasdiluted with 95% HCO₂ H (270 ml). During the addition of HCO₂ H, aprecipitate of 5,6,7,8-tetrahydrofolic acid was formed, whichredissolved rapidly as the volume of HCO₂ H increased (final pH, 1.1).After standing for 18 hours at room temperature, an inorganicprecipitate (5.6 g) was removed by filtration. The filtrate was treatedwith concentrated HCl (3.5 ml) and evaporated to dryness under reducedpressure at 40°. The resulting solid was washed by stirring with cold 1%ascorbic acid (100 ml), collected by filtration, washed with additional1% ascorbic acid solution, and dried in vacuo over P₂ O₅ : yield, 10.0g. A boron analysis indicated that this sample contained boron.

Anal. Calcd for C₂₀ H₂₂ ClN₇ O₆.0.3H₃ BO₃.2H₂ O: C, 43.96; H, 4.96; B,0.59; Cl, 6.49; N, 14.94. Found: C, 43.78; H, 4.89; B, 0.61; Cl, 6.59;N, 17.70.

The above solid was stirred for one hour in cold 0.5 N HCl (100 ml)containing mercaptoethanol (0.1 ml), recollected by filtration under N₂pressure, washed in the funnel with additional 0.01 N HCl (100 ml), anddried in vacuo over P₂ O₅ : yield, 8.4 g (79%). A boron analysisindicated that this sample contained a trace amount of a boron impurity(found, 0.06%).

Anal. Calcd for C₂₀ H₂₂ ClN₇ O₆.H₂ O: C, 47.11; H, 4.74; Cl, 6.95; N,19.23. Found: C, 47.34; H, 4.73; Cl, 6.84; N, 19.29.

(C) A solution of THF.1.66HCl.0.74H₂ O.1.68H₃ BO (2.4 g, 3.9 mmol) in a1:2 mixture of H₂ O-(EtO)₃ CH (60 ml) containing ascorbic acid (0.25 g)was refluxed for five hours and allowed to stand at room temperature for18 hours. The resulting mixture was diluted with (EtO)₃ CH, and thesolid was collected by filtration, washed with Et₂ O, and dried in vacuoover P₂ O₅ to give crude (5,10-CH-THF)⁺ Cl⁻ : yield, 1.5 g. TLC (Avicel,0.1 M phosphate, pH 7) showed that the product was contaminated withfluorescent impurities and ascorbic acid.

EXAMPLE 6 Purification of 5-CHO-THF (Citrovorum Factor) (A) SephadexG-10 Column Chromatography

A glass column (5×118 cm) was poured in one portion with Sephadex G-10(825 g) in H₂ O and packed to a height of 108 cm with H₂ O adjusted topH 8 (meter) with CaO. A solution of impure 5-CHO-THF.Ca (9.0 g) inaqueous Ca(OH)₂ (pH 8) (40 ml) was applied to the column over a periodof four hours, and the resulting column was developed with the samesolvent at a rate of about 16 ml/hour (unless otherwise noted). Afterabout 48 hours, a mixture of 10-CHO-FA and 10-CHO-DHF, which trailedback into the band containing 5-CHO-THF, was eluted. The front of theband containing 5-CHO-THF was visibly yellow. Fractions were taken every30 minutes, and the presence of 5-CHO-THF in a fraction was determinedby TLC on Avicel plates (0.1 M NaH₂ PO₄, pH 7). The combined fractionswere adjusted to about pH 7.5 with aqueous Ca(OH)₂ and diluted with EtOHto the point of cloudiness. After cooling in an ice bath (unlessotherwise noted), the first crop was collected by filtration under N₂. Asecond crop was obtained from the filtrate by the addition of 5 volumesof EtOH. The first experiment involved three column runs, the results ofwhich are summarized in Table 1. On alternate weeks, a second sample of5-CHO-THF was chromatographed on the same column, the results of whichare also shown in Table 1.

The results in Table 1 indicated that p-aminobenzoylglutamic acid iseluted toward the end of the band containing 5-CHO-THF. Also, thisimpurity is concentrated in the (B) samples, which were obtained byintentionally diluting the filtrate with a large volume of EtOH torecover as much weight as possible. In addition, the results indicatedthat elution of most of 10-CHO-FA and 10-CHO-DHF occurred near the frontof the band containing 5-CHO-THF. A greater concentration of 10-CHO-DHFis found in the (A) samples, indicating that the solubility of the Casalt of 10-CHO-DHF is less than the Ca salt of 5-CHO-THF.

One sample (0.82 g) in Table 1 (run I, column 3) was retained. The last3 samples from both run I, column 3, and run II, column 2, werecombined, and the composite sample (6.3 g) was dissolved in H₂ O (125ml). This solution was adjusted to pH 7.5 (meter) with aqueous Ca(OH)₂and diluted with EtOH (˜15 ml) to give a slightly cloudy solutioncontaining a small amount of trash. During the filtration (under N₂) ofthis mixture under aspirator pressure, a yellowish precipitate began todeposit from the unfiltered portion.

                                      Table 1.sup.m                               __________________________________________________________________________              Number of                                                                     fractions               Percent.sup.d                                         (30 min. each)               10-CHO--FA                                       combined Wt..sup.b                                                                              A.sup.282 /A.sup.242c                                                                    and                                    Experiment                                                                              (vol., ml)                                                                             Recovered (g)                                                                          max min                                                                             PABGA                                                                              (5.10-CH--THF).sup.+                                                                   10-CHO--DHF                                                                           5-CHO--THF            __________________________________________________________________________    Crude 5-CHO--THF                                                                        --       --       3.43  <3   <1       <10     ˜86             Run 1, Col. 1                                                                            8 (120)  A. 2.04.sup.e                                                                         3.04  1.3  2.7      <25.6   ˜72             Crude 5-CHO--THF   B. 1.51  3.94  <1   <1       <8.5    ˜90             (9 g) in 40 ml of                                                             solvent   16 (240) A. 1.79  3.99  2    trace    <6.4    ˜92                                B. 1.98  4.59  2    trace    <2.5    ˜95                                total 7.32 (81%)                                           Run 1. Col. 2                                                                           11 (105)  A. 109.sup.f                                                                          3.46  trace                                                                              2.3      <10.4   ˜87             3-CHO--THF (6.4 g) B. 1.24  4.31  trace                                                                              <1.0     <4.0    ˜95             from Col. 1 in 30 ml                                                          of solvent                                                                               9(86)   A. 0.56  4.26  trace                                                                              <1.0     < 5.4   ˜94                                B. 0.95  4.52  2.4  --       <2.3    ˜95                       15 (143) A. 0.90  4.40  trace                                                                              <1.0     <2.2    ˜97                                B. 0.75  4.61  1    --       <1      ˜98                                total 5.48 (86%)                                           Run 1. Col. 3                                                                            10 (78)  A. 0.08.sup.g                                                                         4.22  trace                                                                              <1       <6.5    ˜92             5-CHO--THF (4.2 g) B. 0.82  4.59  trace                                                                              --       <3.8    ˜96             from Col. 2 in 20 ml                                                                    18 (140)  A. 0.08.sup.g                                                                         4.54  trace                                                                              --       <2.5    ˜97             of solvent         B. 2.04  4.58  1    --       <1.3    ˜98                       4 (32)   A. --.sup.g                                                                            --    --   --       --      --                                       B. 0.21  4.73  --   --       <1      ˜99                       3 (24)   A. --.sup.g                                                                            --    --   --       --      --                                       B. 0.18  4.69  --   --       <1      ˜99                                total 2.40 (81%)/ ˜99                                                   total 2.40 (8190)                                          Run B. Col. 1                                                                            12 (94)  A. 1.45.sup.h                                                                         3.43  <1   1.7      < 17.6  ˜80             Crude 5-CHO--THF   B. 2.18  4.14  <1   <1       <7      ˜91             (9 g) in 40 ml of                                                                        12 (94) A. 1.13  4.16  1.1  --       <5.4    ˜93             solvent            B. 1.09  4.50  8.0  --       <1.6    ˜90                        10 (78) A. 0.41  4.65  <1   --       <3      ˜96                                B. 0.67  4.54  2.2  --       <1      ˜97                                total 6.93 (77%)                                           Run B. Col. 2                                                                            7 (53)   A. 0.01.sup.g                                                                         --    trace                                                                              3.4      <22     ˜75             5-CHO--THF (5.4 g) B. 0.53  4.04  trace                                                                              1.1      <9.4    ˜89             in 25 ml of solvent                                                                     14 (105)  A. 0.17.sup.g                                                                         4.35  trace                                                                              <1       <5.6    ˜93                                B. 1.63  4.61  <1   --       <3.1    ˜96                       26 (195)  A. 0.32.sup.g                                                                         4.44  <1   <1       <2.5    ˜95                                B. 1.99  4.52  4.3  --       <1.3    ˜94                                total 4.67 (80%)                                           __________________________________________________________________________     .sup.a The Sephadex G10 column was washed with aqueous Ca(OH), (5.000 ml)     between each run.                                                             .sup.b A refers to the first crop, B to the second crop.                      .sup.c Determined in 0.1 N NaOH, Lederle's 5CHO--THF, ratio 4.86.             .sup.d Amount of impurities estimated by HPLC, 5CHO--THF determined by        difference and does not take into account the percent of solvates and         calcium. Also, small peaks of unidentified substances are observed.           .sup.e Reprecipitated from H.sub.2 O with EtOH to give 1.1 g (ratio           3.36), which was included in sample used for column 2.                        .sup.f Excluded from column 3.                                                .sup.g The cooled mixture was allowed to rewarm to room temperature, whic     redissolved most of the precipitate.                                          .sup.h Excluded from column 3, run 11.                                   

The solid was collected by filtration and dried in vacuo over P₂ O₅ :yield, 0.70 g (11% recovery). TLC (Avicel, 0.1 M NaH₂ PO₄, pH 7) showedthat this sample was mainly 5-CHO-THF contaminated with 10-CHO-DHF,5,10-CH-THF)⁺, and a yellow fluorescent impurity near the origin. HPLCindicated the presence of PABGA (<1%), 10-CHO-DHF (<2.9%),(5,10-CH-THF)⁺ (<1%), and 5-CHO-THF (95.1% by difference). λ_(max) ²⁸²/λ_(min) ²⁴² =4.50.

The filtrate was diluted with additional EtOH (total, 250 ml), and thewhite precipitate was collected by filtration under N₂ : yield, 4.8 g(76% recovery). TLC showed that the sample contained trace amounts of10-CHO-DHF and a yellow fluorescent impurity near the origin. HPLCindicated the presence of PABGA (<1%), 10-CHO-DHF (<1%), and 5-CHO-THF(˜98% by difference). λ_(max), nm (ε×10⁻³): pH 7-286 (30.4); 0.1 NNaOH-282 (29.7). λ_(max) ²⁸² /λ_(min) ²⁴² =4.67.

Anal. Calcd for C₂₀ H₂₁ N₇ O₇.2H₂ O.Ca: C, 43.87; H, 4.60; N, 17.91; Ca,7.32; Ash (CaO), 10.24. Found: C, 43.91; H, 4.63; N, 17.80; Ca, 7.10;Ash (CaO), 10.64.

The filtrate from the above sample was diluted with additional EtOH(total, 625 ml), and the white solid was collected by filtration underN₂ : yield, 0.32 g (5% recovery). TLC showed only 5-CHO-THF. HPLCindicated the presence of PABGA (4.3%) and 5-CHO-THF (95.7% bydifference). λ_(max) ²⁸² /λ_(min) ²⁴² =4.65.

The total amount recovered was 5.8 g (92%).

Purified samples of the calcium salt of 5-CHO-THF gave a λ_(max) ²⁸²/λ_(min) ²⁴² value between 4.6-5.4, probably because a slight change inthe minimum resulted in a large change in the ratio.

(B) Florisil Chromatography

Florisil (350 g, 100-200 mesh) was suspended in H₂ O (3 times), and thefines were removed by decantation. The defined slurry of florisil waspoured into a glass column (3.8×67 cm) and washed with H₂ O until theeffluent was clear and then with 0.2% aqueous mercaptoethanol (4000 ml).After a solution of impure 5-CHO-THF (3.0 g) in 0.2% aqueousmercaptoethanol (10 ml) was applied, the column was developed with 0.2%aqueous mercaptoethanol at a rate of 0.3 ml/min. The progress of thedevelopment was followed by a uv monitor. After 17.5 hours auv-absorbing material, probably p-aminobenzoylglutamic acid, was elutedfrom the column over the next 8 hours. At this time (25.5 hours),5-CHO-THF began to elute and four fractions were collected (see Table2).

                                      Table 2                                     __________________________________________________________________________         Flow rate                                                                           Collection                                                                          Volume,                                                                             Volume 25%.sup.a                                                                        Sample                                       Fraction                                                                           ml/min                                                                              time, hr                                                                            ml    CaCl.sub.2, ml                                                                          wt, g     80.sub.max.sup.282 /λ.su                                               b.min.sup.242                      __________________________________________________________________________    1    0.6   6.5   ˜234                                                                          2         0.36      4.38                               2    0.6   10    ˜360                                                                          3         1.2       4.62                               3    3.0   8     ˜1,400                                                                        2         0.50      3.86                               4    0.6   16    ˜576                                                                         .sup.b                                                       5.0   5     ˜1,500                                                                        4         0.18      2.84                                                             total                                                                            2.24 (75% recovery)                          __________________________________________________________________________     .sup.a After concentration of the fraction in vacuo.                          .sup.b Eluates combined.                                                 

Each fraction was concentrated to about 1/5 volume in vacuo (oil pump)at ˜45° when an off-white flocculent solid began to precipitate. Each ofthe resulting mixtures was adjusted to pH 12 (meter) with 1 N NaOH andfiltered through a thin Celite pad to remove the somewhat gelatinousinsoluble material (MgO). The clear filtrates were adjusted to pH 7(meter) with dilute HCl, treated with 25% aqueous CaCl₂ solution(clarified by filtration), readjusted to pH 7.5 (meter), and dilutedslowly with 5 volumes of cold EtOH. The white precipitates werecollected by filtration and dried in vacuo over P₂ O₅.

In earlier work on florisil chromatography of crude 5-CHO-THF samples ata faster flow rate, the blue fluorescent spots above and below 5-CHO-THF(TLC) were eluted before and during the elution of 5-CHO-THF rather thanafter 5-CHO-THF as described above. Although florisil might have betterabsorbent characteristics for 10-CHO-FA and 10-CHO-DHF at the slowerrate, other work suggested that under these conditions the basic natureof the mobile phase (pH ˜8.5) resulted in the decomposition of theseimpurities.

The same from fraction 1 appeared to contain a water-insoluble materialand was reprecipitated from an aqueous solution with EtOH: yield, 0.29 g(6.5% from FA); λ_(max) ²⁸² /λ_(min) ²⁴² =4.37. HPLC indicated that thissample contained only trace amounts of uv-absorbing impurities.

Anal. Calcd for C₂₀ H₂₁ N₇ O₇.3.8H₂ O.Ca: C, 41.42; H, 4.97; N, 16.91;Ca, 6.91; Ash (CaO), 9.67. Found: C, 41.36; H, 4.91; N, 16.74; Ca, 7.04;Ash (CaO), 9.78.

The sample from fraction 2 (1.2 g, 27% from FA) contained only traceamounts of uv-absorbing impurities (HPLC) and was analyzed withoutfurther purification.

Anal. Calcd for C₂₀ H₂₁ N₇ O₇.3.5H₂ O.Ca: C, 41.81; H, 4.91; N, 17.07;Ca, 6.98; Ash (CaO), 9.77. Found: C, 41.85; H, 4.85; N, 16.90; Ca, 6.93;Ash (CaO), 10.17.

The total yield of practically pure 5-CHO-THF was 1.49 g (33.5% fromFA).

Fraction 3 gave a sample that was shown by HPLC to contain the followinguv-absorbing components: p-aminobenzoylglutamic acid (<1%), 10-CHO-DHF(<5.5%), 10-CHO-FA (˜1%), and 5-CHO-THF (˜92.5% by difference).

The sample from fraction 4 showed (HPLC) the following components:p-aminobenzoylglutamic acid (<2%), 10-CHO-DHF (<20%), 10-CHO-FA (˜1%),and 5-CHO-THF (˜77% by difference).

In a large-scale synthesis, folic acid (600 g) was converted to theimpure calcium salt of 5-CHO-THF via the catalytic hydrogenation of10-CHO-FA over Pt in CF₃ CO₂ H. Four glass columns (8×120 cm) containingFlorisil (2.6 kg) were prepared as described above. The 5-CHO-THF sample(465 g) was divided into 16 portions, and each portion (24-30 g) waseluted (rate, 3-5 ml/min) from one of the columns, each column beingused four times. The development of a column was followed by determingthe HPLC chromatogram of the eluate. During the first run, the 5-CHO-THFband appeared after a volume of 5 l. was collected. In later runs on thesame column, a volume of 7.5 l. was collected before 5-CHO-THF startedto elute. Each 5-CHO-THF fraction (˜4.5 l.) was treated as describedabove. The combined samples were extracted with O₂ -free H₂ O andfiltered to remove an insoluble material. The filtrate was lyophilizedto give the product: yield, 213 g (28% from FA). The HPLC chromatogramshowed only trace amounts of p-aminobenzoylglutamic acid and 10-CHO-DHF.λ_(max), nm (ε×10⁻³): pH 7-287 (31.5); 0.1 N NaOH-282 (30.8). λ_(max)²⁸² /λ_(min) ²⁴² =4.8.

(C) Nonchromatographic Purification of Citrovorum Factor

(1) The hygroscopic, brown, gummy residue (˜400 g) containingglass-fiber filter pads obtained in the conversion of (5,10-CH-THF)⁺ Cl⁻to 5-CHO-THF.Ca was stirred under N₂ in deaerated H₂ O (4 l.), and thepH of the medium was adjusted to 7.5 with solid Ca(OH)₂ (˜1 g). Theglass fibers were removed by filtration, and the residue was washed withportions of deaerated H₂ O (2 l.) until the washings were colorless. Theclear filtrate was cooled in an ice bath and diluted with 0.1 volume ofEtOH (600 ml). The precipitated dark hygroscopic yellow-brown solid wascollected by filtration, washed with EtOH, and dried in vacuo over P₂ O₅: yield, 199 g. λ_(max) ²⁸² /λ_(min) ²⁴² =3.7

The filtrate was diluted with additional EtOH (total, 3 l.), and theprecipitated pale yellow solid was collected by filtration, washed withEtOH and dried in vacuo over P₂ O₅ : yield, 17 g. λ_(max) ²⁸² /λ_(min)²⁴² =3.90.

The clear, yellow filtrate was diluted with additional EtOH (total, 15l.); after cooling, the resulting cream-colored precipitate wascollected by filtration, washed with EtOH, and dried in vacuo over P₂ O₅: yield, 75 g. λ_(max) ²⁸² /λ_(min) ²⁴² (0.1 N NaOH)=4.19. HPLC assay ofthis sample indicated the presence of 5-CHO-THF.Ca (85%), PABGA.Ca(˜2%), 10-CHO-DHF.Ca (˜2%), 10-CHO-FA.CA (trace), and pterins (˜1%). Nodoubt this sample also contained EtOH and H₂ O (<10%).

(2) Additional experiments were carried out on portions of the largecrop of impure 5-CHO-THF obtained as described under procedure (C-1)above.

A turbid solution of this material (3.00 g) in H₂ O (100 ml) containingabout an equimolar amount of MgCl₂.6H₂ O (1 g) was treated portionwisewith solid Ca(OH)₂.. The pH increased rapidly to ˜10.5, then remainedbetween 10.5-10.8. During this period a yellow-brown granularprecipitate separated from the mixture after which the pH increasedrapidly to 12 as more Ca(OH)₂ was added. After stirring for anadditional 30 minutes, the solid was removed by filtration under N₂pressure and dried in vacuo over P₂ O₅ : Yield, 1.40 g. The majorportion of this residue is probably composed of CaO and MgO; however,the HPLC chromatogram showed the presence of a number of impurites andsome 5-CHO-THF.

The clear filtrate was adjusted to pH 7.5 with dilute HCl followed bythe dropwise addition of EtOH (˜20 ml) until permanent turbidity wasreached. This mixture was cooled to 10°, and the yellow solid wascollected by filtration and dried in vacuo over P₂ O₅ : yield, 0.64 g.λ_(max) ²⁸² /λ_(min) ²⁴² =3.71. The HPLC chromatogram showed that thissample contained only small amounts of the usual impurities, butincreased amounts of FA and an unidentified material with a longerretention time. Apparently FA and the unidentified material weregenerated during the base treatment of this sample in the presence ofMgCl₂. A magnesium analysis indicated the presence of a trace amount ofmagnesium (0.003%).

Anal. Calcd for C₂₀ N₂₁ N₇ O₇.Ca.0.5C₂ H₆ O.1.2H₂ O: C, 45.35; H, 4.78;N, 17.63; Ca, 7.21; Ash (CaO), 10.08. Found: C, 45.11; H, 5.01; N,17.38; Ca, 7.20; Ash (CaO), 10.07.

The filtrate was diluted with three volumes of EtOH (300 ml), and theresulting mixture was cooled in an ice bath. The white solid wascollected by filtration, washed with EtOH, and dried in vacuo over P₂ O₅: yield, 1.33 g. λ_(max) ²⁸² /λ_(min) ²⁴² =4.78. HPLC assay andelemental analysis indicated the presence of 5-CHO-THF. Ca (86%),PABGA.Ca (1%), 10-CHO-DHF (<1%), 10-CHO-FA (<1%), pterins (<1%), EtOH(4.2%), H₂ O (3.3%), and unidentified material (˜2.5%).

Anal. Calcd for C₂₀ H₂₁ N₇ O₇.Ca.0.5C₂ H₆ O.H₂ O: C, 45.65; H, 4.74; N,17.75; Ca, 7.25; Ash (CaO), 10.14. Found: C, 45.59; H, 5.06; N, 17.69;Ca, 7.51; Ash (CaO), 10.51.

When the above experiment was repeated using twice the weight of MgCl₂,a smaller amount (0.77 g) of purified 5-CHO-THF was recovered.

It is thought that the invention and many of its attendant advantageswill be understood from the foregoing description, and it will beapparent that various changes may be made in the methods as describedherein without departing from the spirit and scope of the invention orsacrificing its material advantages, the forms hereinbefore describedbeing merely the preferred embodiments thereof.

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
 1. A method for producing tetrahydrofolic acid from folic acid and isolating same, comprising the steps of:(a) reducing folic acid with sodium borohydride in alkaline aqueous solution employing an amount of said sodium borohydride at least equal to the weight of said folic acid to convert said folic acid to tetrahydrofolic acid; (b) adding acid to neutralize the reaction mixture so as to decompose any excess sodium borohydride; (c) dissolving ascorbic acid as an antioxidant in the reaction mixture; (d) acidifying the reaction mixture to a pH sufficient to precipitate said tetrahydrofolic acid; and (e) recovering said tetrahydrofolic acid from the reaction mixture. 